PROJECT SUMMARY: ?Deep characterization of the biogenesis and function of Ebola virus microRNAs? Ebola virus (EBOV) belongs to the group of hemorrhagic fever viruses and causes a severe disease in humans with case fatality rates ranging from 40 to 90%. The devastating EBOV epidemic in West Africa claimed at least 11,000 lives and resulted in billions of dollars of economic loss, highlighting the need for early diagnostic and effective countermeasures. miRNAs are a family of small non-coding RNAs that regulate gene expression at post-transcriptional level6. Interestingly, recent in silico predictions identified EBOV-encoded microRNAs (miRNAs)1-3. However, the functional relevance of these viral miRNAs remains elusive. Recent preliminary data from our labs validated the expression of two published EBOV miRNAs, EBOV- pre-miR-13 and EBOV-pre-miR-T21. However, the small RNA read support and the secondary structures of these loci are incompatible with canonical miRNA processing. Moreover, the 5?-ends of these mature miRNAs coincide with the transcription start site of the EBOV genes VP24 and VP40, respectively. The goal of this project is to uncover the processing pathways involved in the maturation of EBOV-pre- miR-1 and EBOV-pre-miR-T2, and their function during infection. We hypothesize that these miRNAs are processed by a non-canonical miRNA biogenesis pathway that bypasses Drosha cleavage and instead relies on transcription initiation to define the 5?-ends of the miRNAs. We will further explore if the EBOV miRNAs are the product of abortive transcription by the EBOV RNA-dependent RNA polymerase. To gain mechanistic insight into the function of these EBOV-encoded small RNAs, we propose to examine whether EBOV-pre-miR-1 and EBOV-pre-miR-T2!repress host gene expression via the canonical miRNA- effector pathway, or alternatively, if they interfere with EBOV mRNA expression by steric blocking of the transcription start sites on the EBOV genome. Two highly integrated aims will address these hypotheses using a combination of state-of-the-art miRNA analysis and virological approaches. Specifically, we will use the zebrafish embryo and EBOV infection platforms to determine the biogenesis of EBOV-pre-miR-1 and -T2 (Aim 1). In Aim 2, we will perform reporter assays to determine their cellular and viral targets. Finally, we will use modified antisense oligos to interfere with EBOV miRNA function and examine their impact on host and viral gene expression during infection. With the synergy of an interdisciplinary team that combines expertise in microRNAs, zebrafish, bioinformatics and BSL-4 level virology work, we will mechanistically probe the origin and function of EBOV miRNAs. The results from this project will transform our understanding of how small RNAs regulate EBOV infection and inform antiviral drug development to improve the standard of care for EBOV-infected patients. !